Mycobacterial infection is associated with granuloma formation in which the presence of apoptosis has been recognized. The role of CD4+ T and CD8+ T cells in host protection against mycobacterial infections has been demonstrated. Previous studies, however, have shown that CD8+ T cells have a limited role in host defense against Mycobacterium avium infection, and we hypothesize that M. avium infection could lead to T cell apoptosis. To investigate this hypothesis, C57BL/6 mice were infected with M. avium strain 101, and the rate of apoptosis of splenic lymphocytes cultured ex vivo with peritoneal macrophages was determined and compared with that of controls. When exposed to infected macrophages ex vivo, splenic lymphocytes from M. avium-infected mice underwent apoptosis, as determined by the TUNEL assay. This increased T cell apoptosis above the control level was observed after 3 weeks but not after only 1 week of infection in mice. No splenic T cell apoptosis was observed when lymphocytes from Mycobacterium smegmatis-infected mice were cultured in the presence of M. smegmatis-infected peritoneal macrophages. Likewise, macrophages infected in vitro with heat-killed M. avium did not trigger T cell apoptosis. Culture of macrophages in different chamber from lymphocytes, separated by a transwell membrane, was not associated with increase of apoptosis compared with uninfected control, suggesting a requirement for direct cell-cell interactions to trigger lymphocyte apoptosis. Using a double staining TUNEL followed by anti-mouse CD4 or anti-mouse CD8 monoclonal antibodies, it was observed that only CD8+ T cells but not CD4+ T cells underwent apoptosis at 3 weeks of infection. In conclusion, M. avium infection in C57/BL6 mice for 3 weeks renders CD8+ T cells prone to apoptosis when exposed ex vivo to macrophages infected with M. avium.